1. Field of the Invention
This invention relates to novel methods and compositions for retrofitting DNA in single-copy or low-copy vectors with the capability of replicating at higher copy number by contacting the DNA in vitro or in vivo with an artificial transposon containing a conditional multi-copy origin of replication (“ori”).
2. Prior Art
Bacterial Artificial Chromosome (BAC) vectors are now widely used in sequencing projects (Roach, J C, Siegel, AF, van den Engh, G, and Hood, L. “Gaps in the Human Genome Project. Commentary, Nature 401, 843–845, 1999; Ross-McDonald, P, et al. “Large-Scale Analysis of the Yeast Genome by Transposon Tagging and Gene Disruption.” Nature 402, 413–417, 1999; Mahairas, G G, et al., “Sequence-Tagged Connectors: A Sequence Approach to Mapping and Scanning the Human Genome. Proc. Nat. Acad. Sci 96: 9739–9744, 1999; Birren, B, Mancino, V, and Shizuya, H. “Bacterial Artificial Chromosomes.” In: Genomic Analysis: A Laboratory Manual. Volume 3, pp. 241–295, 1999. Cold Spring Harbor Press. Cold Spring Harbor, N.Y.; Frangeul, L, Nelson, K E, Buchreiser, C, Danchin, A, Glaser, P, and Kunst, F. “Cloning and Assembly Strategies in Microbial Genome Projects.” Microbiology 145, 2625–2634, 1999) because they can be used to clone and stably maintain high molecular weight DNA in E. coli host cells. BAC libraries containing genomic DNA inserts of up to about 300 kb are useful tools for generating sequence data. While BAC cloning has no doubt improved the ability to sequence and analyze large genomes, in certain cases BAC sequencing presents significant technical challenges. The prime remaining challenge and still a significant disadvantage of working with BAC DNA is the difficulty of obtaining adequate yields of BAC DNA from small (3 ml) cultures, a feat that is difficult at best. The low copy number of BACs (1–2 copies per host cell) yields only small quantities of DNA, which hampers sequencing projects by decreasing throughput and DNA purity compared to clones in high-copy vectors. BAC DNA can be isolated from larger amounts of culture, but this is more laborious and increases cost and time. Also, for high throughput applications, it is desirable to reduce BAC-containing culture volume below 5 ml in order to facilitate automated purification of BAC DNA. Problems encountered with BAC clones also apply to clones in fosmids and other low-copy vector cloning systems.
Inducible genes have been studied for many years and have been exploited extensively in molecular cloning processes, but have not been studied extensively as a means for at-will control of the copy number of episomal DNA in bacteria. Recently, a system was disclosed for inducing amplification of BACs (Wild, J, et al. Gene 223, 55–66, 1998; Hradecna, Z., et al., Microbial and Comp. Genomics, 3, 58, 1998; U.S. Pat. No. 5,874,259) In these disclosures, the inventors adapted a mutant version of the trfA gene product, the control element for an origin of replication called oriV from plasmid RK2, to an inducible system under the control of the araB promoter. Induction of a mutant trfA gene using the araB promoter enabled replication and amplification of BACs containing oriV.
Other inventors have used an inducible promoter, including an araB promoter, in order to express a protein which is downstream of (i.e., 3′ of) the promoter on a replicable vector (E.g.s, U.S. Pat. Nos. 5,028,530; 6,242,219; and 6,274,344). However, an inducible promoter is used in the compositions and methods of the present invention to express a protein from a gene on a host chromosome and is not used to express a gene on a replicable vector.
Modifying pir-containing E. coli strains so that the wild-type or a mutant pir gene is under the control of an inducible promoter also offers the possibility of at-will control of episomes containing the R6Kγ ori. The R6Kγ ori was originally used for controlling the copy number of extrachromosomal elements in bacteria, depending on the strain of E. coli host bacteria employed (Pillarisetty, Va., et al., “The replication initiator “Pi” of plasmid R6K specifically interacts with the host-encoded helicase dnaB.” Proc. Nat. Acad. Sci. (USA), 93, 5522–5526, 1996). It has found application in the construction of non-viral vectors for DNA vaccines (Suter, M, et al. “BAC-VAC, A Novel Generation of DNA Vaccines: A Bacterial Artificial Chromosome (BAC) Containing a Replication-Dependent, Packaging-Defective Virus Genome Induces Protective Immunity Against Herpes Simplex Virus 1.” Proc. Nat. Acad. Sci. (USA) 96, 12697–12702, 1999). Hansen, et al. (Hansen, L H, et al. “Chromosomal Insertion of the Entire Escherichia coli Lactose Operon into Two Strains of Pseudomonas, using a Modified Mini-Tn5-Delivery System.” Gene 186, 167–173, 1997) recently described the use of the R6Kγori in Tn5-based mini-transposons for moving the lac operon from E. coli into Pseudomonas species.
Recently, several laboratories have developed novel in vitro and in vivo transposon insertion systems. For example, Goryshin and Reznikoff showed that a hyperactive mutant form of Tn5 transposase catalyzes random transposition of an artificial transposon comprising almost any DNA into any other DNA in vitro so long as the artificial transposon has at its termini properly-oriented 19-basepair Mosaic End (ME) sequences that are recognized by the transposase (Goryshin, I. Y., and Reznikoff, W. S., J. Biol. Chem., 273, 7367, 1998). Further work also demonstrated that, in the absence of magnesium cations, one can make a stable synaptic complex between a hyperactive Tn5 transposase and an artificial Tn5 transposon (Goryshin, I. Y., et al., Nat. Biotechnol., 18, 97, 2000). This complex, which has been designated a “Transposome™ complex (EPICENTRE), can be electroporated into living cells, where the intracellular magnesium cations activate the transposase and generate random transposon insertions into cellular DNA in vivo. Various additional aspects of this system are disclosed in U.S. Pat. Nos. 5,925,545; 5,948,622; 5,965,443; and 6,159,736, incorporated herein by reference. Additional information is also available in the 2001 and subsequent editions of the catalog of EPICENTRE Technologies Corporation, Madison, Wis., incorporated herein by reference, and on-line at www.epicentre.com under the heading of “Transposomics™ & EZ::TN™ Transposon Tools,” also incorporated herein by reference. In vitro systems have also been described for other transposons and in vitro transposon insertion kits are commercially available based on the bacteriophage Mu system (Finnzymes, Invitrogen) and on the Tn7 system (New England Biolabs, Inc.).
The advantage of using the Tn5-based Transposome™ system to deliver a R6Kγ ori rather than other Tn5-based systems is that under defined conditions, a single transposon insertion event occurs on either chromosome or extrachromosomal DNA. That is, since the artificial transposons used with Transposome™ systems or other EZ::TN™ insertion systems, as described in the 2001 and subsequent editions of the EPICENTRE catalog, do not encode a transposase gene, the inserted artificial is stable in the insertion site.
It is known that BACs exist stably in only one or two copies in host bacteria (Ross-McDonald, P., et al. “Large-Scale Analysis of the Yeast Genome by Transposon Tagging and Gene Disruption.” Nature 402, 413–417, 1999; Tao, Q. and Zhang, H. “Cloning and stable maintenance of DNA fragments over 300 kb in Escherichia coli with conventional plasmid-based vectors.” Nucleic Acids Res., 26, 4901–4909, 1998). This is an inherent disadvantage for using BACs directly as sequencing templates due to the small amount of DNA obtainable from small cultures. Tao, et al. (ibid) reported that using RK2-based plasmid vectors to increase the copy number of large clones (>300 kb) in an E. coli DH10B™ (Invitrogen) host permitted the maintenance of 5 to 8 copies per cell. While this system for BAC amplification appeared promising, inconsistent results were reported for cloning of human DNA.
What is Needed in the Art
What is needed in the art are improved vectors, including, but not limited to BAC, fosmid and plasmid vectors, into which DNA can be cloned and maintained in host cells at approximately one copy per host cell, but which can be induced to at least about five copies per cell on demand. Preferably, what is needed are vectors into which DNA can be cloned and maintained in host cells at approximately one copy per host cell, but which can be induced to at least about ten copies per cell. Most preferably, what is needed are vectors into which DNA can be cloned and maintained in host cells at approximately one copy per host cell, but which can be induced to at least about twenty or more copies per cell. What is needed in the art are methods that permit researchers to increase the copy number of clones in single-copy BACs, fosmids, or other low-copy vectors at will. What is needed are improved methods and vectors that permit successful cloning and stable maintenance at approximately one copy per cell of DNA comprising repetitive sequences, or AT-rich or GC-rich sequences, or sequences that are toxic or detrimental for the host cell, including without limitation, sequences that comprise one or more genes that encodes one or more peptides or proteins which is toxic or detrimental for the host cell when expressed. In short, what is needed are improved vectors that permit successful cloning and stable maintenance of difficult-to-clone sequences at approximately one copy per cell, but which can be easily and rapidly induced to higher copy number on demand.
Also needed in the art are improved E. coli host strains that contain a gene which encodes at least one protein required by a multi-copy ori for replication, which gene is expressed from a tightly regulated (i.e., “not leaky”), yet easily inducible transcription promoter. Preferably, under induction conditions, these improved host strains support multi-copy replication of appropriate vectors. Most preferably, under induction conditions to multi-copy replication, clones of different clone size in improved host strains yield approximately similar quantities of DNA.
Also needed in the art are compositions and methods that permit single- or low-copy vectors or clones in such single- or low-copy vectors to be easily and rapidly converted to vectors or clones which are capable of multi-copy replication following induction in a suitable host cell. What is needed are systems for using transposons to insert chemically-inducible origins of replication (ori's) in vitro and in vivo into single- or low-copy vectors or clones in such single- or low-copy vectors. What is needed are systems comprising a transposons with a multi-copy ori and an E. coli host strain having at least one gene that can be induced at will to express a protein that is required by the specific multi-copy ori for replication to occur. What is needed are methods for using transposon systems with inducible multi-copy ori's for facilitating sequencing. What is needed are transposon systems with inducible multi-copy ori's for retrofitting existing single-copy BAC libraries by transposon insertion in vivo or in vitro, making existing clones more amenable for automated, high throughput sequencing.
What is needed in the art are transposon systems with inducible multi-copy ori's for in vitro or in vivo insertion directly into genomic DNA. What is needed are copy-controllable systems for “rescue cloning” of genomic DNA for sequencing.
Objects of the Invention
A primary object of the present invention is to improve cloning by permitting control of clone copy number at-will. Another object of the invention is to improve sequencing, particularly high throughput sequencing, by permitting control of clone copy number at-will, most particularly by permitting control of clone copy number for clones in BAC, fosmid, and plasmid vectors.
Another primary object of the invention is to provide improved methods and vectors that permit successful cloning and stable maintenance at approximately one copy per cell of DNA comprising repetitive sequences, or AT-rich or GC-rich sequences, or sequences that are toxic or detrimental for the host cell, including without limitation, sequences that comprise one or more genes that encodes one or more peptides or proteins which is toxic or detrimental for the host cell when expressed. In short, what is needed are improved vectors that permit successful cloning and stable maintenance of difficult-to-clone sequences at approximately one copy per cell, but which can be easily and rapidly induced to higher copy number on demand.
Another primary object of the present invention is to provide improved vectors having an oriV multi-copy origin of replication in order to improve upon the invention described in U.S. Pat. No. 5,874,2590 and related patent applications, incorporated herein by reference.
Another primary object of the invention is to provide a method for using one or more artificial transposons to randomly insert an inducible multi-copy ori into clones in single-copy or low-copy vectors in vitro or in vivo. Another object of this embodiment of the invention is to generate random transposon insertion clones having primer binding sites for bidirectional sequencing of clones which are too large to sequence with a single set of sequencing reactions. Still another object of this embodiment of the invention is to eliminate the need to subclone clones larger than about one kilobase in size into smaller shatter clones for sequencing. Still another object of this embodiment of the invention is to provide a method to control clone copy number at-will at either about one copy per cell or at multiple copies per cell. An object of this embodiment is to permit stable maintenance of large clones at about one copy per cell, while permitting at-will induction to higher copy number for use in sequencing or other purposes. Another object of the invention is to provide compositions and kits comprising artificial transposons having at least one inducible multi-copy ori and, optionally having at least one selectable marker, for use in carrying out the methods of this embodiment of the invention.
Another object of the present invention is to provide a method for obtaining a suitable host strain having a gene which encodes at least one protein required for replication from the oriV origin of replication, which gene is under the control of an inducible promoter, and which host strain is an improved strain for use in the methods of the present invention and for use in the inventions described in U.S. Pat. No. 5,874,2590. Still another object of the present invention is to provide an improved E. coli strain which expresses a mutant form of the trfA gene product under the control of an inducible araB promoter, which strain provides improved results in the methods of the present invention and improved results for the inventions described in U.S. Pat. No. 5,874,2590, and related patent applications by the same inventors.
Another object of the invention was to use an inducible origin of replication in concert with a Transposome™ complex to increase BAC copy number in a suitable host cell.
Another object was to develop methods for using transposons with inducible ori's, such as, but not limited to, the oriV/trfA or R6Kγ/pir systems, and BAC vector systems using either or both, depending on the results of further experiments.
Another object was to construct chemically-inducible ori's, and then to use these to make a system consisting of copy number-controllable ori-containing transposons and complementary E. coli strains host strains that can be induced to express different levels of an appropriate ori-specific protein. An object of this aspect of the invention was to provide a system that is useful in sequencing projects by retrofitting existing BAC libraries by transposon insertion in vivo or in vitro.
Another object was to provide inducible ori-containing transposon systems that would also be useful for in vitro insertion directly into genomic DNA or, using a Transposome™ system, for in vivo insertion and for “rescue cloning” of genomic DNA for sequencing.
A primary object of the invention was to develop systems for amplification of BAC clones in host cells to improve the yield of BAC DNA from small cultures Another primary object of the invention is to provide improved compositions of an oriV/trfA inducible system with respect to applications described elsewhere (Wild, J, Sektas, M, Hradecna, Z, and Szybalski, W. “Targeting and Retrofitting Pre-existing Libraries of Transposon Insertions with FRT and ori'V Elements for in-vivo Generation of Large Quantities of Any Genomic Fragment.” Gene 223, 55–66, 1998).
Another object is to provide an inducible R6Kγ/pir system by replacing the promoter region of the pir gene from E. coli EC100D™ pir+ and EC100D™ pir-116 by an inducible promoter, including but not limited to, an inducible araB promoter, under the control of a simple non-metabolizable chemical using standard recombinant DNA methods.
Another primary object of the invention is to construct inducible oriV- and R6Kγ-containing transposons, BAC cloning vectors and new E. coli host strains for the stable maintenance of multi-copy BACs.
Another object is to construct transposons containing either the R6Kγ or oriV origins of replication by using a plasmid that is specifically designed for transposon construction, i.e., pMOD-2™ (EPICENTRE), which is a plasmid that contains a ColE1 ori (for growth in a standard cloning host), an ampicillin resistance selectable marker, the outer ends (OEs) required for use with the hyperactive Tn5 transposase, PCR priming sites for amplification of the recombinant transposon, and a multiple cloning site in between the OEs.
An object of this aspect of the invention is to construct new transposons by:
a) inserting an inducible origin of replication into the multiple cloning site of a plasmid comprising the transposon construction vector;
b) Inserting a selectable drug marker [e.g., Kan(sup)R] into said plasmid;
c) transforming the recombinant, transposon-containing plasmid into a standard cloning strain of E. coli; 
d) purifying the transposon-containing plasmid;
e) amplifying the recombinant transposon by PCR using standard methods (O'Mullan, P, “Direct Sequencing of BAC Clones Without Subcloning or Primer Walking.” EPICENTRE Forum 7:4, 1–3, 2000. Published by EPICENTRE, Madison, Wis.), and
f) digesting the excised transposon with restriction enzymes PvuII or PshA1, to generate precise transposon ends.
Another object is to use the resulting transposons containing oriV or R6Kγ ori to introduce the inducible origins into BACs by through in vivo insertion (“Transposome”) techniques.
Another primary object of the invention is to develop new strains of E. coli containing an inducible regulatory gene for activating extrachromosomal origins of replication.
An object of this aspect of the invention is to construct new strains of E. coli carrying either the inducible pir gene or inducible ParaBAD-trfA systems, capable of inducing the function of the R6Kγ or oriV, respectively.
Another object of this aspect of the invention is to construct these new strains by using existing strains of E. coli containing either the ParaBAD-trfA system or inducible promoter/pir gene systems.
Another object of this aspect of the invention is that strain constructed should have the following characteristics:    1. It must contain the origin-specific regulatory gene.    2. The regulatory gene must contain a promoter that may be induced using a simple metabolic compound (such as a carbohydrate) and otherwise remain in a state permitting the stable existence of large BACs.    3. It must not have any active recombination systems, such as recABCD, which could, in some cases, permit any extrachromosomal DNA to integrate into the host chromosome by homologous recombination.    4. The new strain should be based upon existing E. coli strains with suitable genetic pedigrees that permit identification of suitable origins of replication and controlling host-encoded regulatory genes that function as needed.
Still another object of this aspect of the invention is that construction of the strain will be accomplished using standard recombinant DNA technologies to modify existing strains of E. coli containing the pir gene (both wild-type and pir-116 mutant) that have been developed for use with existing R6Kγ-containing transposons.
Another primary object of the invention is to construct new BAC vectors containing inducible origins of replications by modification of an existing BAC vector for use in constructing BAC libraries de novo.
An object of this aspect of the invention is to construct a new, inducible vector from the existing pIndigoBAC-5 vector, a derivative of pBeIoBAC and pIndigoBAC (Birren, B, Mancino, V, and Shizuya, H. “Bacterial Artificial Chromosomes.” In: Genomic Analysis: A Laboratory Manual. Volume 3, pp. 241–295, 1999. Cold Spring Harbor Press, Cold Spring Harbor, N.Y.).
Another object of this aspect of the invention is to construct a new vector by modifying the pIndigoBAC vector to contain either the oriV or R6Kγ ori.
Another primary object of the invention is to amplify BAC DNA using an inducible system.
One object of this aspect of the invention is to develop a method to generate “amplified” BAC DNA in a specific E. coli host cell using chemical induction.
Another object of this aspect of the invention is to provide methods for BAC amplification which involve maintaining the BAC at its normal 1–2 copies per cell until growth in culture reaches mid- to late-logarithmic phase and then to introduce a chemical into the culture medium that triggers the ori-regulating gene to be expressed, thereby activating the inducible ori, and leading to replication and amplification of BAC DNA.
Another object of this aspect of the invention is to provide improved methods for BAC amplification with respect to:                1. level of amplification for different sizes of BACs        2. the time required to amplify BAC DNA in an E. coli host by at least 10-fold        3. the stability of the amplified BAC DNA with respect to deletions, etc        
A primary object of this aspect of the invention is that BACs used in the methods of the invention should lack of deletions or other recombination events in order to ensure that a single BAC species is present in a cell.
Another primary object of this aspect of the invention is that at least one of the inducible BAC systems developed can a) maintain a large (>200 kb) BAC in one or two copies until mid- to late-log phase in the host cell, and then be amplified by chemical induction to yield a minimum 10-fold increase in purified BAC DNA over what can be recovered from small cultures (3–5 ml) of a standard BAC using standard BAC DNA isolation methods, such as commercially-available BAC purification systems or standard alkaline lysis procedures (Birren, B, Mancino, V, and Shizuya, H. “Bacterial Artificial Chromosomes.” In: Genomic Analysis: A Laboratory Manual. Volume 3, pp. 241–295, 1999. Cold Spring Harbor Press, Cold Spring Harbor, N.Y.).
Another primary object of the invention is to improve sequence analysis by using amplifiable BACs.
A primary object of this aspect of the invention is to adapt an inducible BAC system to large-scale, high throughput sequencing using automated procedures.
Another primary object of this invention is to develop inducible ori-containing transposons and vectors, as well as complementary E. coli host strains for use in systems for commercialization.
Notations and Nomenclature
The terms used herein have the following meaning with respect to the present invention:
As used herein, a “vector” is a DNA molecule in which other DNA, including, but not limited to “foreign” or “heterologous” DNA, can be operably joined so as to form a circular DNA molecule which can replicate autonomously following its introduction into a host cell. The words “foreign” or “heterologous” refer to the fact that the DNA which is operably joined to the vector is not normally present in the host cell in which it is replicated. The most common method by which the DNA is “operably joined” to a vector is by covalent joining of compatible ends by means of an enzyme referred to as a ligase, such as, but not limited to, T4 DNA ligase, by a process referred to as “ligation.” The process of ligating a DNA molecule in a vector and then replicating this molecule in a host is referred to as “molecular cloning,” and a product of this process is referred to as a “clone.” The methods of the present invention are not limited to a particular vector or host cell, but are intended to apply to any vector and any host in which one can clone and maintain a DNA molecule at a low copy number and then, using the methods of the invention, induce the vector or its clone to a higher copy number whenever desired. By way of example, but not of limitation, a vector of the invention can be a single-copy or a low-copy BAC, fosmid, plasmid, a P1 vector, or nay other suitable vector. The host cells can be E. coli, or any other bacterial or other cells, including prokaryotic or eukaryotic cells, in which the vector is able to replicate at a low copy number, and which can be induced to higher copy number by means of an inducible ori according to the methods of the invention.
As used herein, the word “replicate” refers to the fact that the vector and the DNA to which it is operably joined is copied or reproduced or duplicated in the host cell by a process called “replication.” The site on the vector DNA at which replication begins is referred to as the “origin of replication (“ori”).” An origin of replication which requires the presence of another protein or another molecule in order for replication to occur is referred to as a “conditional origin of replication.” A conditional origin of replication permits one to control replication by providing a means for controlling the expression or properties of the protein or other molecule which is required for replication from the ori. The number copies of a particular vector or of a clone in a particular vector varies based on a different factors, including, but not limit to, the sequence and structure of a particular origin of replication and the structure, amount, and properties of proteins which interact with the origin. For the purpose of the present invention, the oriV disclosed by the inventors comprises the sequence disclosed herein as SEQ. ID NO. 1 and said sequence is preferred in the methods and compositions of the invention. Examples of the present invention include the use of oriV or R6Kγori as an origin of replication, but the methods of the invention are not limited to the use of these ori's, and these same ori's can be modified to some extent to achieve the same effect. Based on the descriptions herein, those with skill in the art will know how to determine whether or not a particular ori is suitable for use in the invention and will be able to identify other ori's for use in the methods and compositions of the invention.
SEQ. ID NO. 1. Sequence of oriV   1GCTGGTTGCC CTCGCCGCTG GGCTGGCGGC CGTCTATGGC CCTGCAAACG CGCCAGAAAC  61GCCGTCGAAG CCGTGTGCGA GACACCGCGG CCGGCCGCCG GCGTTGTGGA TACCTCGCGG 121AAAACTTGGC CCTCACTGAC AGATGAGGGG CGGACGTTGA CACTTGAGGG GCCGACTCAC 181CCGGCGCGGC GTTGACAGAT GAGGGGCAGG CTCGATTTCG GCCGGCGACG TGGAGCTGGC 241CAGCCTCGCA AATCGGCGAA AACGCCTGAT TTTACGCGAG TTTCCCACAG ATGATGTGGA 301CAAGCCTGGG GATAAGTGCC CTGCGGTATT GACACTTGAG GGGCGCGACT ACTGACAGAT 361GAGGGGCGCG ATCCTTGACA CTTGAGGGGC AGAGTGCTGA CAGATGAGGC GCGCACCTAT 421TGACATTTGA GGGGCTGTCC ACAGGCAGAA AATCCAGCAT TTGCAAGGGT TTCCGCCCGT 481TTTTCGGCCA CCGCTAACCT GTCTTTTAAC CTGCTTTTAA ACCAATATTT ATAAACCTTG 541TTTTTAACCA GGGCTGCGCC CTGTGCGCGT GACCGCGCAC GCCGAAGGGG GGTGCCCCCC 601CTTCTCGAAC CCTCCCGG
The examples of the present invention also disclose the use of host cells which express either mutant or wild-type forms of the TrfA Protein, a product of a form of the trfA gene, or of the Pi Protein, a product of a form of the pir gene, as polypeptides which affect replication from oriV or from R6Kγori, respectively. However, the methods of the present invention apply to any ori which can be made conditional on the expression, and the invention can use any protein or other polypeptide or other factor which is required by or which supports an ori that can be used according to the invention in order to obtain multi-copy replication in a desired host.
Preferably, synthesis of a protein or other polypeptide required for replication from a particular ori in the host cells is tightly controlled (i.e., “not leaky”). Most preferably, the protein or polypeptide is controlled by means of an inducible transcriptional promoter that is operably joined “upstream of” or “5′-of” the gene encoding said required protein or polypeptide. A “transcriptional promoter” or more simply a “promoter” is a sequence on a DNA molecule which is recognized by an RNA polymerase enzyme and at which transcription, meaning synthesis of RNA, is initiated.
Transcription of DNA into RNA is required for synthesis and expression of the protein or polypeptide. There are a number of promoters known in the art which are suitable for the methods of the invention. By way of example, but not of limitation, the araB promoter, which can be induced by treating host cells with L-arabinose, and the Tet promoter, which can be induced by treating host cells with anhydro-tetracycline (Lutz, et al., Nucleic Acids Res., 25, 1203, 1997), are preferred promoters in the invention, but there are also many other suitable promoters which can be used. Those with skill in the art will also realize that accessory proteins and the genes which encode them are sometime necessary or beneficial for use in the invention, and are envisioned under the invention. By way of example, but not of limitation, the araC sequence can be linked to an araB promoter for use in the methods and compositions of the invention. As used herein, an “inducer” is a substance that activates the promoter, either by positively regulating the transcription from the promoter, or by binding to a repressor that would otherwise inhibit transcription from the promoter. In either case, the inducer activates transcription from an inducible promoter Transcription of DNA into RNA is required for synthesis and expression of the protein or polypeptide.
A “transposable element”, is a DNA sequence that can move (transpose) from one site in DNA to another.
“Transposition” is the process in which a transposable element is excised from one site and inserted into a second site on the same or another DNA molecule.
A “transposase” is an enzyme that catalyzes transposition. As used herein, the enzyme can be the wild type enzyme or a mutant form of the enzyme, which may, for example, give the enzyme a desirable property, such as, but not limited to, a higher activity. One transposase used in the present invention is a hyperactive mutant form of Tn5 transposase, which is also sometimes referred to as “EZ::TN™ Transposase” (EPICENTRE). However, the invention is not limited to this enzyme, and, unless otherwise specifically limited, other transposases are also intended to be within the scope of and covered by the invention. By way of example, but not of limitation, other transposases which can be used for the methods of the invention include Tn7 transposase, Mu transposase, Mariner transposase, Tn552 transposase (Griffin IV, T J, et al., “In vitro transposition of Tn 552: a tool for DNA sequencing and mutagenesis,” Nucleic Acids Res., 27, 3859–3865, 1999), Tn10 transposase, and the like.
Traditionally, a “transposon” is defined as a transposable element that carries a gene encoding a transposase, as well as a gene or genes with other functions, such as resistance to antibiotics. However, recently Goryshin and Reznikoff (Goryshin, I Y, and Reznikoff, WS, “Tn5 in vitro transposition.” J. Biol. Chem., 273, 7367, 1998) showed that purified wild type and mutant forms of Tn5 transposase can catalyze in vitro transposition of any DNA that is between two properly oriented copies of a Tn5 transposase recognition sequence, which, with respect to Tn5 transposase, is usually called an “Outer End” or an “OE” sequence or an “Inner End” or an “IE” sequence, or a “Mosaic End” or an “ME” sequence, depending on the particular transposase used, but the recognition sequence for any particular transposase which can be used for the invention can also be referred to by a different name. A particular ME sequence was identified which has optimal properties for in vitro transposition using a hyperactive form of Tn5 transposase (Zhou, M A, et al., “Molecular genetic analysis of transposase-end sequence recognition: cooperation of three adjacent base pairs in specific interaction with a mutant Tn5 transposase,” J. Mol. Biol., 276, 913, 1998). In view of the findings of Goryshin and Reznikoff (Goryshin, I Y, and Reznikoff, W S, “Tn5 in vitro transposition.” J. Biol. Chem., 273, 7367, 1998), it will be understood that the gene for the transposase does not need to be present in order to obtain transposition, provided that a transposase is present in a reaction mixture in which the transposase has activity. Therefore, the definition of a “transposon” as used herein, which the inventors sometimes also refer to as an “artificial” transposon, is any DNA that has recognition sequences (or OE sequences or ME sequences) for the transposase such that the DNA is capable of transposase-catalyzed transposition. As used herein, the inventors intend that all transposons of the invention do not encode an active transposase gene (meaning a transposase gene that is expressed in a host cell so as to produce an active transposase), whether or not the transposon is referred to as an “artificial” transposon. It is preferable that a transposon or artificial transposon of the invention does not encode a gene for a transposase because, in the absence of an active transposase gene, the transposon is not be able to transpose to a new location in the absence of added transposase enzyme and suitable reaction conditions.
As used herein, an “EZ::TN™ Transposon” comprises any DNA that transposes into another DNA in the presence of EZ::TN™ Transposase. Any DNA between two properly-oriented 19-basepair transposase recognition sequences or ME sequences recognized by EZ::TN Transposase can serve as an EZ::TN Transposon. A growing number of EZ::TN Transposons having different selectable markers and promoters which are active in different biological systems are available from Epicentre. Examples include EZ::TN <TET-1> Transposon and EZ::TN <KAN-2> Transposon. Alternatively, custom EZ::TN Transposons may be prepared by using a Transposon Construction Vector such as PMOD™ <MCS>, or by PCR using primers containing OE Sequences, or by ligating OE Sequences to the ends of the desired transposon DNA. Although the inventors may refer to EZ::TN Transposons herein, the invention is not limited to these transposons and those with skill in the art will realize that the invention also applies to other transposons or artificial transposons. Specific EZ::TN Transposons will be designated herein as follows: The term “EZ::TN,” which designates an artificial Tn5 transposon having hyperactive ME sequences, is followed by the names of each specific gene within the transposon, each of which is separated from the other by a forward slash (/); then, the names of all of the genes or genetic elements within the transposon are flanked by arrows (< >) which indicate the orientation of the terminal ME sequences; finally, this is followed by the word “Transposon” or “Transposome,” as the case may be. For example, an “EZ::TN <oriV/KAN> Transposon” or an “EZ::TN <or V/KAN> Transposome” designates, respectively, an artificial Tn5 transposon or a Transposome™ complex which has the oriV origin of replication and a kanamycin-resistance gene.
As used herein, an “Insertion Reaction” refers to a reaction which results in transposition of a transposon into a target DNA. EZ::TN Transposase can catalyze insertion of an EZ::TN Transposon into any target DNA in vitro.
A “Transposome™” or a “Transposome™ Complex” as used herein means a synaptic complex formed between a transposon and a transposase, which is stable in the absence of magnesium cations, but which can catalyze insertion of the transposon into another DNA following activation by magnesium cations in vitro or in vivo (Goryshin, I Y, et al., “Insertional transposon mutagenesis by electroporation of released Tn5 transposition complexes”, Nature Biotechnol., 18, 97, 2000; Hoffman, L M, et al., “In vivo transposition of transposon/transposase complexes into the genome of Saccharomyces,” Current Genet., 35, 305, 1999; Hoffman, LM, et al., “Transposome insertional mutagenesis and direct sequencing of microbial genomes,” Genetica, 108, 19–24, 2000). A stable EZ::TN Transposome, which can even be stored for long periods in the freezer, is formed by incubating an EZ::TN Transposon and EZ::TN Transposase in the absence of Mg2+. Some EZ::TN Transposomes, such as EZ::TN <KAN-2>Tnp Transposome, are commercially available from EPICENTRE.
By the “Transposomics™ Field,” the inventors mean a field constituting the myriad in vitro and in vivo applications of Transposomes, whether the Transposome is used as a stable complex or is formed in situ in an in vitro insertion reaction.
“EZ::TN Insertion Kits” or “Transposon Insertion Kits,” as used herein, refer to kits containing reagents and optimized protocols and optionally, controls, for performing in vitro insertion reactions. For example, EZ::TN Insertion Kits for inserting EZ::TN <TET-1> or EZ::TN <KAN-2> Transposons into target DNA in vitro are available from EPICENTRE.
“Transposon Construction Vectors” refer to specially-constructed vectors available from Epicentre for construction of custom EZ::TN Transposons. For example, PMOD™ <MCS> Transposon Construction Vector is a plasmid vector with a multiple cloning site (MCS) between OE Sequences recognized by EZ::TN Transposase.
When used herein, “Deletion/Inversion Vectors” refer to specially-constructed plasmid or cosmid or other circular vectors in which OE sequences are oriented in such a way that transposase-catalyzed transposition results in random unidirectional deletion or inversion of a portion of the DNA that has been cloned into a certain sites on the vector. Examples include the pWEB::TNC™ Cosmid Vector, and pPDM™-1 and pPDM™-2 Deletion Plasmid Vectors.
“Transposon Tools” refers to all kits and reagents which utilize artificial transposons or a transposase.